VIP-related peptides [VIP-related peptides [VIP, PACAP] have widespread effects in the gastrointestinal tract (GI) and central nervous system affecting such diverse functions as motility, secretion and growth of normal and neoplastic tissues. The actions of VIP-PACAP-related peptides is mediated by three receptors (VIP-1-R, VIP-2-R, PACAP-R). Studies in this project are aimed at understanding the molecular basis of action of these different receptors and at developing selective metabolically stable ligands that function as agonists or antagonists at these different receptors. In general, two groups of studies are being performed in this project. To define the pharmacology of VIP receptors, the rat and human VIP-1-R and VIP-2-R receptors have been cloned and stably transfected in CHO and PANC1 cells. Furthermore, various native cells were screened by RT-PCR, Northern blots and binding studies to identify cell lines that have only one native VIP-R subtype. The breast cancer cell line, T47D was found to possess predominantly hVIP-1-R and SupT1 lymphoblasts only hVIP-2-R and to possess sufficient numbers of receptors to be useful for pharmacology and cell biology studies. Using these cells we have demonstrated that the species specific side-effects of a possible therapeutic GRF analogue are due to the selective high affinity interaction of this analogue with hVIP-1-R (Peptides 22:1139-51, 2001). The VIP pharmacophore is being determined for each VIP-R subtype by alanine and D-amino acid screening, to attempt to identify selective ligands. It has now been completed for the rat and human VIP-1-R and is currently being performed in the human and rat VIP-2-R. The analysis of the VIP-1-R demonstrates positions 2,8,9,11,19,24,25,27,28 of VIP can be replaced by alanine with no loss of potency. Furthermore, there are significant differences for the VIP pharmacophore for the VIP-1-R in human, rat and guinea pig that have not been considered in previous analogue design studies. A simplified analogue of VIP with 11 alanines with potency equal to VIP has been made and was found to be metabolically stable. This simplified VIP analogue will be an excellent template for future studies. Furthermore, the pharmacophore of the hVIP-2-R is now almost completed. From these studies simplified selective ligands for the hVIP-1-R and hVIP-2-R can be designed. Secondly, the molecular basis for ligand internalization and the role of cellular cyclic AMP in its mediation by VIP related receptors, is being studied. Receptor mutants with varying lengths of COOH terminus deletions have been made. The effects of mutations in the VIP-1-R and VIP-2-R on internalization of ligand and receptor coupling and affinity for ligands are now being performed.